An important function of the vertebrate kidney is the excretion of potentially toxic chemicals, such as waste products of cellular metabolism, xenobiotics and xenobiotic metabolites. This occurs primarily in the proximal tubule, where renal transport systems remove organic anions and organic cations from peritubular capillaries and transport them across the tubular epithelium into the lumen. Much work has focussed on the mechanisms by which organic anions and organic cations are transported across the surface membranes of the proximal tubular cells, but little attention was paid to the routes by which these compounds cross the cell interior, the assumption being that simple diffusion across the cytoplasm was the only process necessary. We are using comparative renal models (proximal tubules from lower vertebrates and invertebrates and renal cells in culture) in combination with fluorescence microscopy (conventional and confocal), video imaging and intracellular microinjection to test that assumption. We have found that, after transport into renal cells, organic anions are distributed over two compartments: one that is diffuse and cytoplasmic, and a second that is punctate and vesicular. Sequestration in intracellular vesicles is not a result of endocytosis, but rather of uptake from the cytoplasm. Such uptake is concentrative, specific and energy- dependent. These findings indicate that the intracellular distribution of organic anions is not uniform and that, since a substantial fraction of cellular organic anions are sequestered, the average cellular concentrations measured in most experiments greatly overestimate cytoplasmic concentrations. Future studies will focus on: 1) characterizing the mechanism of vesicular organic anion sequestration in renal cells, 2) determining if organic cations are also sequestered and by what mechanism, 3) defining the role sequestration plays in overall renal xenobiotic secretion, and 4) searching for analogous intracellular secretion mechanisms in other specialized epithelia that accumulate and transport organic anions and cations, e.g., choroid plexus.